1. Field of the Invention
The present invention relates to the field of detection of biological agents using novel compositions, methods and apparatus comprising one or more nucleic acid ligands operably coupled to an organic semiconductor. More particularly, the present invention relates to the production and use of nucleic acid ligands against anthrax spores.
2. Description of Related Art
There is a great need for the development of methods, compositions and apparatus capable of detecting and identifying known or unknown chemical and biological agents (herein referred to as analytes), which include but are not limited to nucleic acids, proteins, illicit drugs, explosives, toxins, pharmaceuticals, carcinogens, poisons, allergens, contaminants, pathogens and infectious agents.
As one skilled in the art will readily appreciate, any method, technique or device capable of such detection and identification would have numerous medical, industrial forensic and military applications. For instance, such methods, techniques and devices could be employed in the diagnosis and treatment of disease, to develop new compounds for pharmaceutical, medical or industrial purposes, or to identify chemical and biological warfare agents.
Current methods, techniques and devices that have been applied to identification of chemical and biological analytes typically involve capturing the analyte through the use of a non-specific solid surface or through capture deoxyribonucleic acids (DNA) or antibodies. A number of known binding agents must then be applied, particularly in the case of biological analytes, until a binding agent with a high degree of affinity for the analyte is identified. A labeled antiligand (e.g., labeled DNA or labeled antibodies) must be applied, where the antiligand causes, for example, the color or fluorescence of the analyte to change if the binding agent exhibits affinity for the analyte (i.e., the binding agent binds with the analyte). The analyte may be identified by studying which of the various binding agents exhibited the greatest degree of affinity for the analyte.
There are a number of problems associated with current methods of chemical and biological agent identification. It takes a great deal of time and effort to repetitiously apply each of the known labeled antiligands, until an antiligand exhibiting a high degree of affinity is found. Accordingly, these techniques are not conducive to easy automation. Current methods are also not sufficiently robust to work in the heat, dust, humidity or other environmental conditions that might be encountered, for example, on a battlefield or in a food processing plant. Portability and ease of use are also problems seen with current methods for chemical and biological agent identification.
Within the field of biological warfare, there is a great need for a rapid, sensitive method to detect and identify pathogenic spores of Bacillus anthrax (hereafter xe2x80x9canthraxxe2x80x9d). Anthrax is a highly pathogenic biological agent that is relatively simple to produce and distribute in the field. Present methods for detection of anthrax are not sufficiently rapid, sensitive, and robust to allow early detection of exposure to anthrax under field conditions, such as might be encountered on a battlefield. No good method presently exists for neutralization of anthrax under field conditions.
The present invention fulfills an unresolved need in the art, by providing methods, compositions and apparatus for the production of nucleic acid ligands capable of binding to, identifying and/or neutralizing anthrax. The methods and compositions disclosed herein provide substantial improvements over earlier methods for anthrax detection (e.g., Reif et al., 1994; Gatto-Menking et al., 1995; Bruno and Yu, 1996), by utilizing anthrax-binding nucleic acid ligands.
The compositions of the present invention comprise a recognition complex or a recognition complex system that are capable of detecting, identifying, characterizing or purifying a chemical or biological agent (hereafter, xe2x80x9canalytexe2x80x9d), preparing or purifying high affinity nucleic acid ligands for selected known analytes, using high affinity nucleic acid ligands to measure the concentration of analyte in a sample or to neutralize an analyte, or to perform high through-put screening of libraries of compounds or native plant extracts for compounds that are structural analogs of known inhibitors, activators or binding agents of bioactive molecules. The recognition complex and recognition complex system and the corresponding techniques should be capable of full automation.
Each recognition complex is comprised of a nucleic acid ligand operably coupled to an organic semiconductor. In certain embodiments, the organic semiconductor is DALM (diazoluminomelanin), although the use of other organic semiconductors, such as polyphenylenes, is contemplated within the scope of the invention. In various embodiments, the organic semiconductor may be attached to the nucleic acid ligand by either covalent or non-covalent interaction.
In preferred embodiments, the nucleic acid ligand is DNA, although it is contemplated within the scope of the invention that other nucleic acids comprised of RNA or synthetic nucleotide analogs could be utilized as well. In certain embodiments, the nucleic acid ligand sequences are random, or may be generated from libraries of random DNA sequences. In other embodiments, the nucleic acid ligand sequences may not be random, but may rather be designed to react with specific target analytes. In a preferred embodiment, the nucleic acid ligand sequences are aptamers (Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; 5,843,653; 5,864,026; 5,989,823 and PCT application WO 99/31275, each incorporated herein by reference).
In certain embodiments, the analyte to be identified may be added in the form of a complex mixture that may include, for example, aqueous or organic solvent, proteins, lipids, nucleic acids, detergents, particulates, intact cells, bacteria, viruses and spores, as well as other components. In other embodiments, the analyte may be partially or fully purified before exposure to the array. In particularly preferred embodiments, the analyte is anthrax spore.
In certain embodiments, a recognition complex system, comprising two or more recognition complexes, may be used in methods for identifying an analyte. After the analyte is contacted with the recognition complexes, certain recognition complexes will bind the analyte, while others will not. Binding of analyte to a recognition complex may be detected by changes in the electrochemical properties of the nucleic acid ligand/organic semiconductor couplet upon binding to the analyte. Nonlimiting examples of electrochemical signals include photochemical, fluorescent or luminescent signals, changes in color or changes in electrical conductivity. The degree to which the electrochemical properties change is a function of the degree to which the nucleic acid ligand binds the analyte. Accordingly, the electrochemical changes that occur across all of the recognition complexes, when taken as a whole, can be used as a unique signature to identify the analyte.
To facilitate detection of such electrochemical changes, the recognition complex system may be associated with a detection unit operably coupled to the recognition complexes. Non-limiting examples of detection units include a charge coupled device (CCD), a CCD camera, a photomultiplier tube, a spectrophotometer or a fluorometer. The recognition complex system may also be associated with system memory for storing electrochemical signals, as well as a data processing unit that may comprise a neural network or lookup tables. For embodiments where the binding of analyte is detected by changes in electrical conductivity of the recognition complex, the complexes may be positioned between a pair of electrodes attached to a conductivity meter.
In addition to analyte identification, recognition complexes may be used to screen for the presence or measure the amount of analytes that are biological molecules, such as hormones, cytokines, vitamins, metabolites or other compounds, in samples of human tissue, fluids or extracts. Nucleic acid ligands with high affinity for biological molecules of interest may be prepared as described below. Upon exposure of recognition complexes incorporating the high affinity ligands to a sample, the presence of the biological molecule is indicated by its binding to the ligand. Since binding of analyte to ligand results in an electrochemical signal, the concentration of biological molecule in the sample can be readily determined by quantifying the signal. Where the biological molecule of interest is part of a macromolecular complex, flow cytometry may also be used to detect and quantify the amount of biological molecule in a sample.
In certain embodiments, the recognition complex system may be used to enrich or purify analytes that bind to one or more selected nucleic acid ligands. In a preferred embodiment, selected nucleic acid ligands are attached to a surface and exposed to a population of analytes. After binding of analyte to nucleic acid ligand, the unbound analytes are removed and the enriched or purified bound analyte is eluted from the ligand. Enrichment and purification may occur using either an interative process, with multiple cycles of binding, separation and elution, or by a single-step process. Separation of bound from unbound analyte may occur by any method known in the art. In a non-limiting example, the ligands may be attached to a column chromatography resin or other solid support and exposed to a mixture of analytes. Unbound analyte may be removed by simple washing of the column or other support. Bound analyte may be eluted by exposure to solutions containing appropriate salt concentration, pH, detergent content, chaotrophic agent or other substance that interferes with the binding interaction. Depending on the affinity of analyte for ligand and the stringency of the initial binding interaction, it may be possible to obtain a relatively purified analyte with a single binding step.
In certain embodiments, the recognition complexes may be attached to a surface, such as a Langmuir-Blodgett film, functionalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold, silver, membrane, nylon, glass bead, magnetic bead or PVP. In preferred embodiments, the recognition complex system of the present invention employs organic semiconductor chip technology wherein nucleic acid ligands are distributed across the surface of the chip so as to form an array of recognition complexes. In other embodiments, the recognition complexes of the present invention may be attached to a surface for use in a flow cell apparatus.
In additional embodiments, the nucleic acid ligands are attached to magnetic beads instead of to a chip. An array of nucleic acid ligands may be assembled, each attached to a magnetic bead. In certain embodiments, each nucleic acid ligand attached to a single magnetic bead has the same nucleic acid sequence, while in other embodiments a single magnetic bead may be attached to nucleic acid ligands of different sequences. In a preferred embodiment, the magnetic bead is attached to an organic semiconductor, such as DALM, and the nucleic acid ligand is attached to the organic semiconductor, forming an array of recognition complexes. Although any method may be employed within the scope of the present invention to attach the organic semiconductor to the magnetic bead and the nucleic acid ligand to the organic semiconductor, in a preferred embodiment the organic semiconductor is covalently attached to the magnetic bead and the nucleic acid ligand is non-covalently attached to the organic semiconductor. In a more preferred embodiment, the attachment of nucleic acid ligand to organic semiconductor is an electrostatic interaction, preferably mediated by magnesium ion.
In certain embodiments, an array of recognition complexes attached to magnetic beads is exposed to an analyte and binding of analyte to nucleic acid ligand may be detected, for example, by photochemical changes in the nucleic acid ligand/DALM couplet upon binding to the analyte. The skilled artisan will realize that magnetic beads would be particularly useful for separating recognition complexes that bind to the analyte from recognition complexes that do not bind the analyte. In one embodiment, a magnetic flow cell, such as is described in U.S. Pat. No. 5,972,721 (incorporated herein by reference), could be used in conjunction with the recognition complex system to identify and separate analyte-binding recognition complexes from recognition complexes that do not bind the analyte.
In certain preferred embodiments, flow cytometry is used to separate recognition complexes that bind to an analyte from those that do not bind. In such embodiments, the recognition complex may be attached to a glass or other bead, or the analyte may comprise a population of cells, spores or other large particles for analytical or preparative procedures. Nucleic acid ligands that bind to the target analyte, or analytes that bind to a specific nucleic acid ligand, may be sorted, for example, by screening particles for DALM-associated fluorescence in a flow cytometer.
In certain embodiments, the recognition complex system may be subject to an iterative process to increase the specificity and affinity of the nucleic acid ligands for an analyte of interest. In such embodiments, nucleic acid ligand sequences that bind a selected analyte are identified, separated, amplified (e.g., using a polymerase chain reaction) and attached to organic semiconductor to form a new recognition complex system. The nucleic acid ligand sequences that do not bind to the analyte are discarded. The new recognition complex system is exposed to the analyte and binding of analyte to nucleic acid ligands produces an enhanced electrochemical signature, as the nucleic acid ligand sequences present will more specifically compliment the analyte. This procedure may be repeated, with each iteration producing a more unique or enhanced signature.
In a further embodiment, this iterative process may be used to identify and amplify one or more nucleic acid ligand sequences that exhibit the highest degree of affinity for a specific analyte. Production of a nucleic acid ligand that binds to the analyte with high affinity (dissociation constant of 1.0 xcexcM or lower) would have utility in a variety of applications. For certain embodiments, production of a nucleic acid ligand with a dissociation constant of 100 nM or lower, more preferably 10 nM or lower, most preferably 1 nM or lower is preferred. This process also provides a method for purifying a nucleic acid ligand that binds to a target analyte. Purification may be less than 100%, the only requirement being that the nucleic acid ligand of interest is present in significantly greater proportion in the final mixture compared to the starting material. A xe2x80x9cpurifiedxe2x80x9d nucleic acid ligand may comprise 10% or more, preferably 20% or more, more preferably 40% or more, more preferably 60% or more, more preferably 80% or more, more preferably 95% or more of the total nucleic acid content of the xe2x80x9cpurifiedxe2x80x9d fraction.
It is contemplated within the scope of the present invention that separation of bound from unbound nucleic acid ligands may occur using virtually any method that can separate bound from unbound ligands. Non-limiting examples include use of nucleic acid chips, use of magnetic beads and magnetic filters, use of glass or other beads and flow cytometry, and flow cytometry using cells as the target analyte. In any case, further iterations of the binding and separation steps will result in progressive enrichment (purification) of ligands that bind to the analyte. If desired, the stringency of the binding interaction may be increased, for example by increasing the temperature or by raising or lowering the salt concentration or the pH of the solution.
In another embodiment, nucleic acid ligands that bind to the analyte with high affinity can be reproduced (synthesized or amplified) for use as a neutralizing agent to inactivate or destroy the analyte. A high affinity nucleic acid ligand may be attached to a variety of agents that could be used to neutralize the analyte, such as toxic proteins, enzymes capable of activating protoxins, or other molecules or reactive moieties including radioisotopes and other organic or inorganic compounds. In certain embodiments, the high affinity nucleic acid ligand can be attached to an organic semiconductor, such as DALM. The DALM/nucleic acid ligand couplet, after binding to the analyte, may be activated by a variety of techniques, including exposure to sunlight, heat, or irradiation of various types, including laser, microwave, radiofrequency, ultraviolet and infrared. Activation of the DALM/nucleic acid ligand couplet results in absorption of energy, which may be transmitted to the analyte, inactivating or destroying it. See U.S. Pat. No. 6,303,316, incorporated herein by reference.
In certain embodiments, the high affinity nucleic acid ligand could be incorporated into an apparatus capable of being carried into the field. For example, the high affinity nucleic acid ligand could be incorporated into a patch or card to be worn by an individual. Exposure of the individual to the specific analyte for which the nucleic acid ligand exhibits high affinity could be indicated by a color change of the patch, or by a change in the electrical or photochemical properties of a nucleic acid ligand/organic semiconductor couplet. Alternatively, the high affinity nucleic acid ligand could be incorporated into an apparatus to be carried by a vehicle that could be used to cover a wide area to detect and identify unknown chemical or biological agents.
The skilled artisan will realize that the scope of the present invention is not limited to applications in chemical or biological warfare, but rather includes a broad variety of potential applications in industry and medicine, where early detection and identification of exposure to chemical or biological agents is desired. Non-limiting examples of such applications include to detect explosives or illegal drugs in an airport detection system, to detect air-borne pathogens in an air conditioner monitoring system, to detect water-borne pathogens, carcinogens, teratogens or toxins in a water quality monitoring system, to detect pathogens in a hospital operating room monitoring system, to screen for pathogens in samples of human tissues or fluids, to detect allergens, pathogens or contaminants in a food production monitoring system, to detect genetically modified organisms, or to perform high through-put screening for pharmaceutical compounds.